Mach 3 rabbit hrp polymer detection

Remaining alginate slabs were either fixed in formalin and paraffin‐embedded or frozen for immunohistochemical evaluation. Sections of the slabs were stained for insulin (guinea pig polyclonal; dilution 1:400; Fitzgerald, Acton, MA), detected by MACH 3 Rabbit HRP‐Polymer Detection (Biocare Medical, Concord, CA) and visualized by 3,3′‐diaminobenzidine.
Alginate‐embedded formalin‐fixed slabs and islets from the pancreas donors were stained with Congo red according to Puchtler and Sweat,12 and the presence of amyloid was determined in polarized light.13Biopsies from the tissue surrounding the device were obtained at explantation and fixed in formalin. Sections (5 μm) were stained for CD31 (mouse monoclonal, clone JC70A), CD4 (mouse monoclonal, clone 4B12), CD8 (mouse monoclonal, clone C8/144B), CD3 (rabbit polyclonal, code A0452), CD20cy (mouse monoclonal, clone L26), CD45 (mouse monoclonal, clone 2B11 + PD7/26), and CD68 (mouse monoclonal, clone KP1). Antibodies were purchased from Agilent Technologies (Kista, Sweden).

Paraffin-embedded islet grafts (n = 8) were consecutively sectioned, and every other section was stained for insulin (guinea pig polyclonal; dilution 1:400; Fitzgerald, Acton, MA), detected by MACH 3 Rabbit HRP-Polymer Detection (Biocare Medical, Concord, CA) and visualized by 3,3′-diaminobenzidine.^{36 (link)} The insulin area was determined manually using a Leica LMD6000-microscope (Leica Microsystems, Wetzlar, Germany). The total area of the islet graft was calculated as an area under the curve by plotting the known areas.
Sections were double-stained for insulin (Fitzgerald) and glucagon (mouse monoclonal, dilution 1:800; Abcam, Cambridge, UK) with Alexa Fluor 488 (goat anti-guinea pig) and Alexa Fluor 594 (donkey antimouse; dilution 1:250; Invitrogen, Carlsbad, CA) as secondary antibodies. Images were acquired with Zeiss LSM780 (Zeiss, Jena, Germany) confocal. The area of insulin and glucagon was determined with the image software Imaris (Bitplane AG, Zurich, Switzerland). The estimated total α cell area was calculated by multiplying the α cell percentage with the total insulin area for the respective islet graft.